Mutations in the NXF-1:NXT-1 mRNA export complex affect gene-expression driven by the hsp-16.41 promoter

The NXF-1 : NXT-1 heterodimer is essential for the nuclear export of mRNA. Here we describe three new alleles of nxf-1 and one allele of nxt-1 isolated from a forward genetic screen. These mutations cause no apparent phenotype under normal growth conditions, but partially suppress the lethality caused by heat-shock induced expression of the PEEL-1 toxin from P hsp-16.41 :: peel-1 . There is also decreased expression of P hsp-16.41 ::eGFP in an nxf-1 mutant. We propose that NXF-1 : NXT-1 influences the expression of heat-shock activated genes due to a role in the recruitment of the hsp-16.41 promoter to the nuclear pore complex during heat-shock.

(A) Schematic of NXF-1 and NXT-1 indicating functional domains (see text for details) and the mutations that were identified in our screen. (B-C) Quantification of ::peel-1 lethality as the fraction of moving (living) worms at different times after a 2 hr heat shock at 34°C; n=3-5, each replicate represents 50 animals; error bars represent standard error of the mean. (D) Schematic of expected outcomes and quantification of dead progeny from animals heterozygous for the zeel-1(tm3419) deletion and homozygous for nxf-1(yak65) with endogenously expressed peel-1. WT control shows quantification of dead progeny from animals heterozygous for zeel-1(tm3419). n=7-9, each n represents 12-127 animals; ns -not significant (p>0.05); error bars represent standard deviation. (E) Relative fluorescence of ::GFP in an nxf-1(yak65) mutant normalized to control; n=3 replicates, each replicate group represents 8-17 animals; bars represent means. Statistics: unpaired two-tailed t tests.
PEEL-1 is a toxin found in the sperm of C. elegans which is lethal to embryos lacking the antidote ZEEL-1, and PEEL-1 is also toxic to adult worms when expressed ectopically (Seidel et al., 2008(Seidel et al., , 2011. The mechanism of this lethality is not yet known. To better understand how PEEL-1 kills, we performed a genetic screen for suppressors of the lethality caused by peel-1 expression driven by the heat-shock promoter hsp-16.41. Among the mutants found in this screen were four recessive partial suppressors. Three mutants (yak63, yak64, and yak65) were mapped to chromosome V, and one to chromosome I (yak135). By whole-genome sequencing we found that yak63, yak64, and yak65 are missense mutations in nxf-1 and that yak135 is a missense mutation in nxt-1 ( Figure 1A).
We quantified the partial-suppression of the nxf-1 and nxt-1 mutants by performing a heat-shock time-course experiment. Expression of Phsp-16.41::peel-1 was induced by heat shock, and death was measured by response to touch. All wild-type control animals died within the first three hours, while more than 80% of nxf-1 and nxt-1 mutant animals remained alive ( Figure 1B,C). At 24 hours after heat shock, survival ranged from 42-92%. Notably, nxf-1(yak63) conferred the least resistance to Phsp-16.41::peel-1, and this allele is the only one of the four found outside of any known functional domain.
To confirm that nxf-1(yak65) affects the expression of heat-shock induced genes, we generated nxf-1(yak65) animals expressing GFP under the hsp-16.41 promoter. The average fluorescence of these worms upon heat-shock was reduced by 72% compared to worms with wild-type nxf-1 ( Figure 1E), further supporting the idea that nxf-1 affects expression from the hsp-16.41 promoter.
Because NXF-1:NXT-1 appears to specifically affect expression of genes driven by the hsp-16.41 promoter, the NXF-1:NXT-1 complex appears to be acting at the level of the DNA in contrast to its more widely described role in exporting mRNA. A plausible mechanism is that NXF-1:NXT-1 may be involved in recruiting the hsp-16.41 promoter to nuclear pores for efficient transcription and export of mRNA driven by this promoter. In C. elegans, localization of the bidirectional heat-shock promoter hsp-16.2/41 at the nuclear pore complex was shown to occur during heat-shock (Rohner et al., 2013). This localization depended on a component of the TREX-2 complex, which was proposed to act as a tether between the hsp-16.2/41 promoter and the nuclear pore complex (Rohner et al., 2013). Interestingly, the human TREX-2 component GANP directly interacts with NXF1 (Wickramasinghe et al., 2010), suggesting that TREX-2 and NXF-1:NXT-1 may together recruit the hsp-16.2/41 promoter. However, we note that two of our nxf-1 alleles (yak64 and yak63) are located in N-terminal regions of this protein, while a C-terminal fragment of human NXF1 carrying just its NTF2 and UBA domains was sufficient for binding to GANP (Wickramasinghe et al., 2010).
The nxf-1 and nxt-1 mutants we isolated are overtly wild-type. By contrast, loss-of-function mutations or RNAi knockdown of nxf-1 and nxt-1 cause severe defects in mRNA export and lethality (Tan et al., 2000;Zheleva et al., 2019), suggesting that our mutations may specifically perturb recruitment of the hsp-16.41 promoter without affecting the general mRNA export functions of NXF-1:NXT-1.

Methods
Strain maintenance: C. elegans worms were maintained at 20°C or room temperature (~23°C) on NGM agar plates seeded with lawns of OP50 bacteria (Brenner, 1974).
Suppressor screen: Strains XZ1174 and XZ1372, which both contain two single-copy insertions of Phsp-16.41::peel-1, were mutagenized with ENU or EMS as described (Brenner, 1974;De Stasio & Dorman, 2001). F2 animals were heat-shocked at 34° for two hours, and survivors were isolated. yak63 was isolated from EMS mutagenesis, while yak64, yak65, and yak135 were isolated from ENU mutagenesis. All of these mutations are recessive.
Gene identification: Mutations were mapped to chromosomes using marker strains EG8040 and EG8041 (Frøkjaer-Jensen et al., 2014). yak63, yak64, and yak65 were mapped to chromosome V, and yak135 was mapped to chromosome I. A complementation test suggested that yak64 and yak65 belong to the same gene. yak63, yak64, and yak65 were outcrossed five times, and yak135 three times using strain XZ1047 to generate strains XZ63, XZ64, XZ65, and XZ135. Genomic DNA was isolated from these strains using the Hobert lab protocol found at http://hobertlab.org/wpcontent/uploads/2013/02/Worm_Genomic_DNA_Prep.pdf. Whole-genome sequencing was performed by Novogene and the data were analyzed using the Galaxy web platform (version 18.09) at https://usegalaxy.org/. Mutations were confirmed by Sanger sequencing. The nxf-1 mutations are as follows: yak63 is a C to T mutation leading to P286S, yak64 is a C to T mutation leading to L101F, and yak65 is a G to A mutation leading to R486K. The nxt-1 mutation yak135 is a T to G mutation leading to I75S.
Heat-shock: 50 gravid adults of strains XZ63, XZ64, XZ65, and XZ135 along with controls XZ1174 or XZ1372 were placed on new plates at 34°C for two hours and then moved to 20°C or room temperature. Animals were probed gently by worm pick, and response to touch was measured either as movement or no movement. Animals were assayed immediately following heatshock and then every thirty minutes for three hours, with a final measurement at 24 hours. This experiment was performed a minimum of three times for each strain.
Phsp-16.41::GFP fluorescence assays: Strains XZ2418 and XZ2457 carrying a single-copy insertion of Phsp-16.41::eGFP::H2B were heat-shocked for 2 hours at 34°C. Animals were then placed at room temperature for 2 hours before mounting to agarose slides with 50 mM sodium azide. Entire animals were imaged using a NikonTi2-E Crest X-light V2 spinning disk confocal microscope by excitation at 488 nm and capture with a 510/20 filter. Each image was taken as a Z series with 0.8 μm steps. Images were analyzed using FIJI utilizing the Z-project sum slices tool (Schindelin et al., 2012). ROIs were drawn around each animal and measured for mean gray value. Background was subtracted from these values using the same ROIs placed outside of the animal. Control worm mean-gray values after background subtraction were averaged for each replicate and used to normalize all values for that replicate.   III;